In the mammalian testis, spermatogenesis starts from a small population of spermatogonial stem cells (SSCs) that self-renew and differentiate to ultimately produce sperm. Failure of any of the molecular events underlying spermatogenesis will result in non-obstructive azoospermia in men. Non-obstructive azoospermia affects approximately 1% of men. Upon testis biopsy, azoospermia presents with Sertoli cell-only pattern, maturation arrest (MA), or hypospermatogenesis. In studies examining patients with non obstructive azoospermia, approximately 30% of the cases were identified with MA. In two studies that examined only patients with MA, all were found to be deficient in either the Notch-1 receptor or its ligand Jagged. Thus, 1 in 300-400 men may have a form of testicular failure resulting from a deficiency in Notch signaling. However to date, the exact role of Notch signaling in the testis is unknown. The Notch proteins (Notch1-4) are large cell-surface receptors that are activated by contact with membrane-bound ligands on neighboring cells, such as Jagged and Delta (Dll). Upon activation, the Notch intracellular domain (NICD) is cleaved and translocates to the nucleus where it associates with DNA-binding proteins and upregulates the expression of target genes, most notably the Hes/Hey family of transcriptional repressors. Hes/Hey proteins are bHLH transcription factors that form homo or heterodimers to function properly. In addition to interacting with other bHLH proteins, mainly co-repressors, and binding to specific DNA promoter sequences, they also can recruit chromatin modifiers such as histone deacetylases (HDACs). The presence of a number of Notch receptors and ligands in the testis has been established, but to date no functional studies have been attempted to identify their exact role. We have evidence that Jagged1 triggers Notch1 activation and upregulates Hes1 in undifferentiated spermatogonia, while it triggers Notch3 activation and upregulation of HeyL in pachytene spermatocytes, specifically at the XY body. Therefore, the effects of Jagged1 depend on the target cell. In this application, we will test the hypothesis that Notch signaling, through up- regulation of Hes1 and HeyL, plays a crucial role at 2 critical steps of spermatogenesis, 1) spermatogonial differentiation and 2) XY silencing at meiosis. PUBLIC HEALTH RELEVANCE: Spermatogenesis is the process of sperm formation in the testis. It starts with a spermatogonial stem cell that self-renew or differentiate into more mature spermatogonia. These cells develop into spermatocytes that will undergo meiosis to become haploid spermatids and sperm cells. Failure of any of the molecular events underlying spermatogenesis will result in infertility. Recent studies indicate that a number of infertility cases might be linked to a defect in the Notch signaling pathway. Notch is a large transmembrane receptor at the surface of germ cells, which receives signals from the somatic nursing Sertoli cells. This proposal seeks to understand the function of Notch and its downstream intracellular targets at 2 critical steps of spermatogenesis: spermatogonial differentiation, and meiosis. )